Recently, it has been shown that introduction of double-stranded RNA (dsRNA) also called interfering RNA (RNAi), or hairpin RNA is an effective trigger for the induction of gene silencing in a large number of eukaryotic organisms, including animals, fungi, and plants.
Both the qualitative level of dsRNA-mediated gene silencing (i.e., the level of gene silencing within an organism) and the quantitative level (i.e., the number of organisms showing a significant level of gene silencing within a population) have proven superior to the more conventional antisense RNA or sense RNA mediated gene silencing methods.
For practical purposes, the production of antisense RNA molecules and chimeric genes encoding such antisense RNA is more straightforward than the production of dsRNA molecules or the genes encoding those RNA molecules. Indeed, the chimeric nucleic dsRNA molecules or the genes encoding those RNA molecules contain large, more or less perfect inverted repeat structures, and such structures tend to hamper the intact maintenance of these nucleic acids in intermediate prokaryotic cloning hosts. The methods and means to increase the efficiency of antisense-RNA mediated gene silencing as hereinafter described provide a solution to this problem as described in the different embodiments and claims.
U.S. Pat. No. 5,190,131 and EP 0 467 349 A1 describe methods and means to regulate or inhibit gene expression in a cell by incorporating into or associating with the genetic material of the cell a non-native nucleic acid sequence. The sequence is transcribed to produce an mRNA that is complementary to and capable of binding to the mRNA produced by the genetic material of that cell.
EP 0 223 399 A1 describes methods to effect useful somatic changes in plants by causing the transcription in the plant cells of negative RNA strands which are substantially complementary to a target RNA strand. The target RNA strand can be an mRNA transcript created in gene expression, a viral RNA, or other RNA present in the plant cells. The negative RNA strand is complementary to at least a portion of the target RNA strand to inhibit its activity in vivo.
EP 0 240 208 describes a method to regulate expression of genes encoded in plant cell genomes, achieved by integration of a gene under the transcriptional control of a promoter which is functional in the host. In this method, the transcribed strand of DNA is complementary to the strand of DNA that is transcribed from the endogenous gene(s) one wishes to regulate.
WO95/15394 and U.S. Pat. No. 5,908,779 describe a method and construct for regulating gene expression through inhibition by nuclear antisense RNA in mouse cells. The construct comprises a promoter, antisense sequences, and a cis-or trans-ribozyme that generates 3′-ends independently of the polyadenylation machinery and thereby inhibits the transport of the RNA molecule to the cytoplasm.
WO98/05770 discloses antisense RNA with special secondary structures such as (GC)n-palindrome-(GC)n, or (AT)n-palindrome-(AT)n, or (CG)n-palindrome-(CG)n, and the like.
WO 01/12824 discloses methods and means for reducing the phenotypic expression of a nucleic acid of interest in eukaryotic cells, particularly in plant cells, by providing aberrant, possibly unpolyadenylated, target-specific RNA to the nucleus of the host cell. Unpolyadenylated target-specific RNA may be provided by transcription of a chimeric gene comprising a promoter, a DNA region encoding the target-specific RNA, a self-splicing ribozyme and a DNA region involved in 3′ end formation and polyadenylation.
WO 02/10365 provides a method for gene suppression in eukaryotes by transformation with a recombinant construct containing a promoter, at least one antisense and/or sense nucleotide sequence for the gene(s) to be suppressed, wherein the nucleus-to-cytoplasm transport of the transcription products of the construct is inhibited. In one embodiment, nucleus-to-cytoplasm transport is inhibited by the absence of a normal 3′ UTR. The construct can optionally include at least one self-cleaving ribozyme. The construct can also optionally include sense and/or antisense sequences to multiple genes that are to be simultaneously downregulated using a single promoter. Also disclosed are vectors, plants, animals, seeds, gametes, and embryos containing the recombinant constructs.
Zhao et al., J. Gen. Virology, 82, 1491-1497 (2001) described the use of a vector based on Potato Virus X in a whole plant assay to demonstrate nuclear targeting of Potato spindle tuber viroid (PSTVd).
WO 02/00894 relates to gene silencing methods wherein the nucleic acid constructs comprise within the transcribed region a DNA sequence that consists of a stretch of T bases in the transcribed strand.
WO 02/00904 relates to gene silencing methods wherein nucleic acid constructs comprise (or encode) homology to at least one target mRNA expressed by a host, and in the proximity thereto, two complementary RNA regions which are unrelated to any endogenous RNA in the host.